CN102286431B - Monoclonal antibody for resisting Japanese encephalitis virus (JEV) and application thereof - Google Patents
Monoclonal antibody for resisting Japanese encephalitis virus (JEV) and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biology, and relates to a monoclonal antibody for resisting the Japanese encephalitis virus (JEV) and application thereof. The hybridoma cell strain 2B4 which can excrete the monoclonal antibody for resisting the JEV is preserved in the China General Microbiological Culture Collection Center on August 3, 2011, and the preservation number is CGMCC No. 5124. The monoclonal antibody for resisting the JEV is produced by the hybridoma cell strain 2B4 with the preservation number of CGMCC No. 5124. The monoclonal antibody can specifically identify the JEV, and both cell and animal experiments show that the monoclonal antibody has neutralizing activity and can be applied in the preparation of detecting reagents for detecting the JEV. The successful preparation of the anti-JEV monoclonal antibody lays a foundation for the further research on the JEV, the establishment of a faster and more sensitive and convenient detection method and the development of related diagnostic reagent kits, test paper and vaccines.
Description
Technical field
The invention belongs to biological field, relate to monoclonal antibody and the application thereof of anti-encephalitis b virus.
Background technology
(Japanese encephalitis virus JEV), belongs to the flaviviridae Flavivirus, and tunicary sub-thread positive chain RNA virus, structural protein have 3 kinds: C, M and E are divided into 4 genotype (I, II, III, IV type) to encephalitis b virus.Culex tritaeniorhynchus is main communication media, mainly popular in the Asia, the main popular gene I type of China and III type, it is one of most important arboviruses of harm China's people's life and livestock industry, can cause serious neurological symptom, lethality rate is up to about 30%, and other has 50% patient can stay nonvolatil neural system sequela.According to statistics, the annual encephalitis number of the infected in Asia reaches about 16000 examples, and about dead 5000 examples, China is except Xinjiang, Tibet, Qinghai, and all there is morbidity other each province with popular, and encephalitis morbidity number accounts for the world's more than 80% of number of always falling ill.In recent years, this sick epidemic regions becomes serious public health problem in continuous expansion.It is very important strengthening monitoring and diagnosing.
At present, the encephalitis diagnostic method mainly contains serodiagnosis, separation and Culture and molecular biology method.
1, serology detects:
Traditional serological method has hemagglutination-inhibition test (HI), complement fixation test (CFT) (CT), neutralization test (NT) etc.At present main use euzymelinked immunosorbent assay (ELISA) (ELISA) arranged, immunofluorescence (IFA), drip golden immunization.Hemagglutination-inhibition test, complement fixation test (CFT), neutralization test are epidemiology survey traditional methods commonly used, need to gather acute phase and decubation paired sera detect, diagnosis is wasted time and energy, can not reach the purpose of early stage quick diagnosis, specificity and susceptibility are not high, and the collection of double sample is had any problem; The development of enzyme linked immunological coating technique, enzyme labelling technology, monoclonal antibody technique has promoted application and the development of ELISA detection encephalitis IgM.Now be applied to diagnose the ELISA method of encephalitis IgM that prize law and indirect method are arranged; Immunofluorescence technique operation more complicated needs personnel and the equipment of specialty, and drips golden immune rule because it is simple, fast, is expected to promote produce and uses.
2, separation and Culture:
Mainly containing suckling mouse brain and organize inoculation and cellular segregation such as BHK, Vero, is to obtain the basis that strain carries out molecule epidemic disease-ology research and vaccine strain structure.Obtain strain by separation and Culture, carry out evaluations such as molecular biology, can study its genotype, the whether main epidemic strain in this area or external input strain, whether morph etc.
3, molecular Biological Detection
Owing to have serological cross reaction widely between flavivirus, morbidity early part patient does not also produce antibody, makes encephalitis serodiagnosis can not reach promising result; The encephalitis patient toxicaemia phase is very short, and the viral separation and Culture time is long, and positive rate hangs down the generation of RT-PCR technology and early stage, quick, responsive, special method is provided for the virus detection.The development of progress, molecular biology and information biology that JEV complete genome sequence mensuration obtains, provide may for the rapid gene of encephalitis detects.Main detection method has PCR-ELISA microplate hybrid method, RT-PCR, sleeve type PCR, half sleeve type PCR, real-time fluorescence PCR and gene chip.
From 1975, Kohler and Milstein founded hybridoma technology.The Monoclonal Antibody technology applies to the every field of biology and medical science widely because of the development of the humanization technology of its very strong specificity and specificity, especially monoclonal antibody.In the detection of encephalitis b virus, hybridoma cell technology and monoclonal antibody technique play the important and pivotal role at aspects such as ELISA, neutralization test, immunofluorescence, immune colloid gold reagent box, display technique of bacteriophage.The monoclonal antibody of neutrality then has great potential aspect the development of vaccine and detection kit.
Summary of the invention
The purpose of this invention is to provide a kind of hybridoma of secreting the monoclonal antibody of anti-JEV.
Another object of the present invention provides the monoclonal antibody of anti-JEV.
Another purpose of the present invention provides the application of this monoclonal antibody.
Secrete the hybridoma cell strain 2B4 of the monoclonal antibody of anti-encephalitis b virus, on August 3rd, 2011 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.5124.
A kind of monoclonal antibody of anti-encephalitis b virus is the hybridoma cell strain 2B4 generation of CGMCC No.5124 by described preserving number.
The application of described monoclonal antibody in the detection reagent of preparation detection encephalitis b virus.Described monoclonal antibody in the exo-antigen antibody response for detection of the neutralizing effect in cause of disease, viral protein and host cell and the experimentation on animals.For example be used for the neutralization experiment of enzyme-linked immunosorbent assay (ELISA), western blotting (Western Blot), indirect immunofluorescence (Indirect Immunofluorescence), cell and animal and the detection that flow cytometry (Flow Cytometry Assay) waits, to detect it with associativity, the specificity of antigen and whether to have the activity of neutralization.
The monoclonal antibody of the anti-JEV that the present invention makes, for the research of JEV with set up new encephalitis b virus detection method and relevant diagnostic kit and vaccine provides favourable instrument.
Beneficial effect:
The JEV virus that this paper utilizes the BHK-21 cell cultures to go out, concentrate and the sucrose density gradient centrifugation purifying through ultracentrifugation, as the antigen immune BALB/C mice, through conventional cytogamy, screening and cloning, set up the hybridoma cell strain (2B4) of the anti-JEV monoclonal antibody of stably excreting.Through the evaluation of methods such as ELISA, Western Blot, the secreted monoclonal antibody of hybridoma can be identified encephalitis b virus specifically, and cell and experimentation on animals show that all this monoclonal antibody has the neutralization activity.
The successful preparation of the anti-JEV monoclonal antibody of the present invention is for the further research of JEV and set up diagnostic kit, test strip and vaccine quicker, sensitive, that detection method and exploitation easily is correlated with and lay a good foundation.
The application of the anti-JEV monoclonal antibody of the present invention:
(1) this antibody can detect the level of the JEV in cerebrospinal fluid and various body fluid such as blood plasma, lymph liquid, serum etc.
(2) utilize this monoclonal antibody to carry out experimentation on animals.
(3) can utilize monoclonal antibody to carry out pharmaceutical research, be used for the treatment of the encephalitis B that causes with JEV.
Description of drawings
The RT-PCR detected result of each layer sample after Fig. 1 sucrose density gradient centrifugation,
Wherein swimming lane 1 is the sample of sample application zone one deck absorption, and swimming lane 2 is the sample that 45%-60% one deck is drawn, and swimming lane 3 is the sample that 30%-45% one deck is drawn, and swimming lane 4 is the sample that 30%-sample application zone one deck is drawn.
JEV virus particle under Fig. 2 electron microscopic observation,
Wherein "-" all represents 100nm, the sample that A draws for 45%-60% one deck, the sample that B draws for 30%-45% one deck.
Fig. 3 monoclonal antibody 2B4 respectively dilutes the fluorescence picture of gradient,
A is monoclonal antibody 2B4 dilution 10
-1The fluorescence picture, B is monoclonal antibody 2B4 dilution 10
-2The fluorescence picture, C is monoclonal antibody 2B4 dilution 10
-3The fluorescence picture, D is monoclonal antibody 2B4 dilution 10
-4The fluorescence picture, the positive contrast of P, the negative contrast of N.
Fig. 4 Western Blot detects monoclonal antibody 2B4 and bhk cell lysate and the cytotoxic specific reaction of JEV, and wherein swimming lane 1 is bhk cell lysate+monoclonal antibody 2B4, and swimming lane 2 is JEV cell toxicant+monoclonal antibody 2B4.
Fig. 5 flow cytometry monoclonal antibody 2B4 under field conditions (factors) with bhk cell and infect JEV bhk cell in conjunction with situation, A blank wherein, B only adds the negative control that fluorescence two resists, the reaction of C monoclonal antibody 2B4 and bhk cell, the reaction of the bhk cell of D monoclonal antibody 2B4 and infection JEV.
In Fig. 6 cell and the experiment.
Among Fig. 7 and experiment-indirect immunofluorescence.
The indirect immunofluorescence of Fig. 8 monoclonal antibody 2B4 detection of cancerous encephalitis B suckling mouse brain spinal fluid is photo as a result,
Wherein, A～D is followed successively by the gradient dilution (10 that monoclonal antibody 2B4 of the present invention detects 10 times in work
-1, 10
-2, 10
-3, 10
-4) test sample BS-V figure as a result, E is many anti-positive controls (producing with the ordinary method immunize rabbit), and F detects BS figure as a result for monoclonal antibody 2B4 of the present invention, and G is for only adding the anti-negative control of fluorescence two.
Biological sample preservation information
Hybridoma cell strain 2B4, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the preservation address is No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCC No.5124, preservation date on August 3rd, 2011.
Embodiment
The pure totivirus antigen of embodiment 1 preparation
The propagation of step 1 virus
(Shanghai biological company limited of brilliant section) is incubated at and contains 8% calf serum (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.) with bhk cell, the penicillin of 100U/ml, among the DMEM of 100 μ g/ml Streptomycin sulphates, outwell nutritive medium after growing up to cell monolayer, wash 1 time with the nutritive medium of PBS (pH7.2) or serum-free, inoculation JEV (SA14-14-2, bank Bioceuticals Inc. in the Hunan), 37 ℃ of effect 1.5-2h, use PBS (NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO4 4.3mmol/L then, KH2PO41.4mmol/L) or the nutritive medium of serum-free wash 1 time, add 2% the liquid of keeping, put 37 ℃ of CO2 incubators and cultivate, microscopically is observed after 2～3 days, when cytopathy (CPE) when reaching 85% ,-20 ℃ frozen stand-by.
Concentrating and purifying of step 2 virus
With the viral multigelation collected 3 times, the centrifugal 20min of suspension 5000r/min, it is stand-by to get supernatant (S1), the PBS with 0.01M suspends again with precipitation, the usefulness ultrasonic treatment it, 100W, 30S/ time, 3 times repeatedly.Get supernatant (S2) in centrifugal 20 minutes with 6000r/min again, discard precipitation.S1 and S2 are mixed, add NaCl and PEG6000, make its final concentration reach 2%-3% and 7% respectively, stir down at 4 ℃ and spend the night, next day, 7000r/min was centrifugal 20 minutes, supernatant discarded, and precipitation TNC (10mM Tris, 10mM NaCl and 2mMCaCl2, pH7.8), damping fluid is suspended into 240ml, again with 27000r/min centrifugal (SW28 rotor) 2 hours, supernatant discarded, suspending with TNC is precipitated to 12ml.This viral suspension is added on gently on 30%, 45%, 60% the non-linear sucrose density gradient, again with the centrifugal 3h of 100000g/min, collects the protein extract on each gradient zones band respectively.Dilute protein extract on each gradient zones band with TNC respectively again, with 27000r/min centrifugal 2 hours, abandon supernatant, with a small amount of TNC precipitation that suspends, frozen stand-by.Step 3RT-PCR identifies and electron microscopic observation
The whole process of this step is all operated at the biologic cleanliness worktable except water-bath and PCR part.
To the protein extract on each collected in embodiment 1 step 2 gradient zones band, i.e. four groups of samples (sample application zone, 30%-sample application zone, 30%-45%, 45%-60%; Each component addition sequence of centrifuge tube is 60% sucrose solution, 45% sucrose solution, 30% sucrose solution, sample; That segment body that adds sample when sample application zone just refers to application of sample is long-pending, gets the sample on adjacent two kinds of different densities sucrose solution interfaces then respectively.) carry RNA, be RT-PCR according to the system of JEV Cap albumen, the identifying virus region, and four groups of samples are carried out electron microscopic observation, process is as follows:
(1)RT-PCR
Every group of sample got 250 μ l, and adds PBS and be diluted to 500 μ l, adds 700 μ l Trnzol (cracking), and gentle mixing leaves standstill 2min up and down.Add chloroform 200 μ l (Deproteinization), leave standstill 2min behind the concuss, the centrifugal 15min of 12000r/min.Get supernatant, and add equal-volume Virahol (precipitated rna), place more than the 15min in-20 ℃.Then, the centrifugal 20min of 12000r/min abandons supernatant, and with 75% washing with alcohol once, fully dries.At last, with 20 μ lRNAse free H
2O is resuspended.
By system (dNTP-4 μ l; Oligo DT-1 μ l; Sample, the RNA-5 μ l that namely extracts in the above-mentioned steps; RNAse free H
2O-3 μ l) added all components, put into 65 ℃ of water-bath 5min after, place 2min on the ice chest again.Then, according to system (5 * Fsbuffer-4 μ l; DTT-2 μ l; RNA enzyme inhibitors-0.5 μ l) added all components, put into 37 ℃ of water-bath 2min, placed ice chest to add 0.7 μ l mouse reversed transcriptive enzyme then, put into the PCR instrument then, 37 ℃ of 50min have been set, 75 ℃ of 15min.
According to the PCR system:
10×Fs buffer 5μl；
dNTP 4μl；
Upstream primer is (SEQ ID NO.1) 1 μ l;
Downstream primer is (SEQ ID NO.2) 1 μ l;
RTag enzyme 0.5 μ l;
RNAse free H
2O 34μl；
Sample 5 μ l
Put into the PCR instrument after having added all components, 95 ℃ of pre-sex change 1min, amplification condition is: 94 ℃ of 1min, 57 ℃ of 1min, 72 ℃ of 1min, 35cycles, 72 ℃ are extended 1min.
At last, run glue and identify (the horizontal strip electrophoresis system is available from Bio Rad company product), as shown in Figure 1, between 30%-45% and 45%-60%, virus is arranged.
(2) electron microscopic observation
4 groups of samples mentioning in embodiment 1 step 3 are carried out electron microscopic observation, result such as Fig. 2, in 30%-45% one deck sample many virus particle are arranged, and content is fewer in 45%-60% one deck sample, and many foreign proteins are arranged, therefore, comparatively speaking, virus in 30%-45% one deck sample is purer, and is standby as immunogen in following examples after the survey concentration.
The foundation of embodiment 2 hybridoma cell lines
With 3 BALB/C mice of the full poison immunity of the encephalitis of the 30%-45% of purifying among the embodiment 1 (6-8 ages in week, available from PLA Military Medical Science Institute Experimental Animal Center): i is immunity for the first time: encephalitis b virus 150 μ g/ are only, add equal-volume good fortune formula Freund's complete adjuvant (CFA, sigma company) fully behind the mixing with 1ml/ subcutaneous multi-point injection BALB/C mice only, 0.2ml/ point, 3 weeks at interval.Ii is immunity for the second time: dosage and approach are the same, and this adds equal-volume good fortune formula Freund (IFA, sigma company), at interval 3 weeks.Iii is immunity for the third time: dosage is the same, does not add adjuvant, uses isopyknic physiological saline instead, the abdominal injection BALB/C mice; Get after 10 days by the eyeball blood of immune mouse (namely put 4 ℃ of refrigerator overnight after the collection, next day with the centrifugal 10min of 2000r/min after, stay the supernatant standby).With the encephalitis b virus bag quilt of purifying, measure serum titer by ELISA.The 4th booster immunization of iv: dosage and approach be with immunity for the third time, get serum titer greater than 1: 10000 mouse in merging preceding 3 days abdominal injections.
Step 2ELISA method detects by the serum titer flow process of immune mouse
Detect the day before yesterday, be cushioned liquid (0.05molL with bag
-1NaHCO
3-Na
2CO
3Damping fluid, pH9.6) encephalitis b virus of dilution purifying (encephalitis of the 30%-45% of embodiment 1 preparation or purifying is poison entirely) adds in the enzyme linked immunological adsorption plate, and every hole 100 μ l put 4 ℃ of refrigerator overnight; Next day is to the interior liquid of enzyme linked immunological absorption plate hole that goes out to wrap quilt, (every 1L PBS adds 0.5ml Tween-20 with lavation buffer solution, PH7.4) washing is totally 3 times, pat dry at every turn, (PBS of every 100ml adds 1gBSA to add confining liquid 150 μ l/ holes, pH7.4, BSA is available from Beijing ancient cooking vessel state Bioisystech Co., Ltd), put 37 ℃ of effects 2 hours; With aforesaid method washing 3 times, pat dry, adding PBST carries out serum to be checked (extent of dilution is respectively: 1: 100,1: 1000,1: 2000,1: 4000,1: 8000,1: 16000) the 100 μ l/ holes of gradient dilution, with the negative contrast of the serum of non-immune BALB/C mice, with antibody titer after the immunity greater than the serum that separates in 1: 10000 the BALB/c mouse eyeball blood serve as the contrast positive serum, put 37 ℃ of effect thermostat containers 1 hour; With the PBST washing, method is the same, pats dry again, and sheep anti-mouse igg antibody (the prosperous Bioisystech Co., Ltd of Beijing ancient cooking vessel state carries out dilution in 1: 20000 with PBST) the 100 μ l that every hole adds the HRP mark put 37 ℃ of effect thermostat containers 1 hour; Wash 3 times, pat dry, it is (now with the current that every hole adds TMB colour developing liquid; Substrate buffer solution is 0.05molL-1PH5.0 phosphoric acid-citric acid; It is the hydrogen peroxide 35 μ l that add 100 μ l TMB and 75% in every 10ml substrate buffer solution that TMB uses liquid, and TMB is available from the prosperous Bioisystech Co., Ltd of Beijing ancient cooking vessel state) 100 μ l, put 37 ℃ of effect thermostat container 15-20min; Add stop buffer (2M H2SO4) 100 μ l/ holes then, detect the OD450nm value with microplate reader, with the blank zeroing, S detects the value in hole, the mean value of the negative contrast of N, the mean value of the positive contrast of P for each.Under the situation of P/N 〉=3, S/N=treats the OD450nm of the OD450nm value/negative control hole of verify, and mean value 〉=3 o'clock are judged to be the positive.
The preparation of step 3 myeloma cell, splenocyte and feeder cell
(1) myeloma cell's preparation
2 all recovery myelomatosis SP2/0 cells (Shanghai Ya Ji bio tech ltd) before fusion, taking out the back in liquid nitrogen container thaws rapidly in 37 ℃ of water-baths, the centrifugal 8min of 1500r/min, abandon behind the supernatant resuspended with 1640 (Gibco company) substratum of 20% foetal calf serum, put 37 ℃, cultivate in the 5%CO2 incubator, change liquid next day, after cultured continuously was treated the activation recovering of myelomatosis oncocyte in several days, culturing bottle with 6 dress 5ml substratum, the myeloma cell of 10 times of serial dilutions of inoculation, quite close and a bottle of not growing remigrates (its objective is in order to make cell be in logarithmic phase) to cell in 1 week back.12h changes liquid once before merging.Merge the same day, cell is blown down from the bottle wall gently, be collected in the 50mL centrifuge tube 1000rmin
-1Centrifugal 5-10min abandons supernatant.Sedimentation cell is with the incomplete substratum of 30mL, once centrifugal with method, be resuspended in then the 10ml serum-free 1640 in mixing.Add the blue staining fluid of 0.4% phenol and make viable count, standby, about 4 * 10
7/ ml.
(2) preparation of splenocyte
Get the positive BALB/c mouse of immunity back antibody titer, extract the eyeball blood sampling, and the positive control serum of separation of serum when being antibody test.Take off neck and put to death mouse, immerse the 5min that sterilizes in 75% alcohol, put into Bechtop, the aseptic abdominal cavity of opening, take out spleen, place the plate that contains incomplete 1640 substratum of 10mL, the reticular tissue on spleen surface is peeled off in washing gently, then spleen is placed in another plate that contains the incomplete RPMI-1640 of 10mL, and place 200 eye mesh screens, and grind gently with plunger, make it discharge splenocyte.With suction pipe piping and druming for several times, make single cell suspension, 1000rmin
-1Centrifugal 5-10min, full substratum centrifuge washing 1-2 time of toing many or too much for use is resuspended in splenocyte in the 50mL V-arrangement centrifuge tube that contains the incomplete RPMI-1640 of 10mL then, add the blue dye liquor of platform phenol do behind the viable count standby, about 2 * 10
8/ ml.
(3) preparation of feeder cell
Preparing feeder cell (peritoneal macrophage) before cytogamy in 1-2 days causes death mouse by above-mentioned method of adopting mouse (BALB/C mice in 6 ages in week) splenocyte, body surface sterilization and fixing after, cut off skin of abdomen to expose peritonaeum with sterile scissors, sterilize with the cotton ball soaked in alcohol wiping, with the incomplete RPMI-1640 of asepsis injector per injection 10ml to the abdominal cavity, flushing repeatedly, the sucking-off washing fluid is put into 50ml centrifuge tube (about 40ml altogether), with the centrifugal 5-10 of 1000r/min minute, with the 5mlHAT substratum sedimentation cell is suspended earlier after abandoning supernatant, according to count results, add 2 * 10
5/ ml adds 96 porocyte culture plates, and 37 ℃ 5% CO is put in 100 μ l/ holes
2Cell culture incubator is cultivated.
A is preheated to 40 ℃ with 50%PEG (pH8.0) 1ml, and incomplete substratum (20-30ml) is preheated to 37 ℃.
B is with 1 * 10
8Splenocyte and 3 * 10
7Individual myeloma cell SP2/0 is mixed in the 50ml fusion pipe, adds incomplete substratum to 30ml, fully mixing.The centrifugal 5-10min of 1000r/min blots supernatant only (very important, as can to influence fusion efficient) as far as possible.And have gentle hands pat beat fusion pipe at the bottom of, make sedimentation cell loose evenly, put preheating in the water-bath in 40 ℃.
C adds the 50%PEG of preheating with the 1ml suction pipe along tube wall about 45s, the limit edged stirs gently.Add the incomplete substratum of preheating along tube wall with the 10ml suction pipe in 90s, 37 ℃ leave standstill 10min, and the centrifugal 5min of 1000r/min abandons supernatant.
D adds the 5mlHAT substratum, and the pressure-vaccum sedimentation cell suspends and mixing it gently, add then contain peritoneal macrophage the HAT substratum to 80-100ml.
E divides and to be filled to 96 porocyte culture plates, and 100-500 μ l/ hole places culture plate 37 ℃ 5% CO
2Cell culture incubator is cultivated.After 5 days with HAT (Sigma company) substratum 1/2 substratum that swaps out.Use HT (Sigma company) substratum affected part HAT substratum after 7-10 days, available common perfect medium after 14 days.
F observes the growth conditions of hybridoma, treat its grow to hole floorage 1/10 when above the sucking-off supernatant for antibody test usefulness.The screening of step 5 hybridoma
Merge and can begin screening about 2 weeks, operate according to embodiment 2 steps 2, difference is encephalitis b virus to the 15 μ g/ml that is cushioned liquid dilution purifying with bag; Adding hybridoma supernatant extent of dilution to be checked is respectively: 1: 100,1: 1000,1: 2000,1: 4000,1: 8000,1: 16000, cloning was just carried out in the high hole of the positive value of result.
The cloning of step 6 hybridoma, enlarged culturing and frozen
The cloning of hybridoma adopts limiting dilution assay to carry out, and concrete grammar is as follows:
A prepares the feeder cell suspension: method is with (3) unanimity in embodiment 2 steps 3.
The hybridoma suspension that b preparation is to be cloned, with the HT substratum that contains 20% serum be diluted to every milliliter contain 2.5,15 with 3 kinds of different extent of dilution of 50 cells, by 5 * 10
4-1 * 10
5The ratio of/ml adds peritoneal macrophage respectively.96 orifice plates of every kind of hybridoma packing, each extent of dilution 32 hole, 200 μ l/ holes, the quantity in every hole is respectively 0.5,3,10.
The CO of c37 ℃ 5%
2Cell culture incubator was cultivated 7-10 days, macroscopic clone occurs and can detect antibody, under inverted microscope, observe, mark the hole of having only single clonal growth, get supernatant and do antibody test, cell in the positive hole is carried out next time cloning, so repeat just to obtain after 3 times can the stably excreting monoclonal antibody hybridoma (all mono-clonal holes of a strain cell cloning next time of carrying out cloning are all positive).
The cell 2B4 that d chooses in the positive the strongest mono-clonal hole changes cultivation (hole is changeed in a hole) in 24 orifice plates over to, in 24 orifice plates, be divided into 2 holes after 3 days, be divided into 4 holes after 3 days again, after 2 days, change the cell in this 4 hole in 50ml Tissue Culture Flask enlarged culturing in the lump again, thereby obtained secreting the hybridoma cell strain of the monoclonal antibody of anti-JEV, deliver the China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, hybridoma cell strain 2B4 preserving number is CGMCC No.5124, preservation date on August 3rd, 2011.
E hybridoma frozen: treat 50ml culturing bottle inner cell be paved with bottle at the bottom of about 70% o'clock, with the cell in the culturing bottle with suction pipe piping and druming to suspending fully, move in the aseptic centrifuge tube of 50ml cell suspension and counting, centrifugal 1200r/min, 6min; Abandon supernatant, (foetal calf serum is available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd. with cells frozen storing liquid (30% foetal calf serum, 60% incomplete RPMI-1640,10%DMSO); DMSO is available from Shanghai Ling Feng chemical reagent company limited) resuspended precipitation, regulating cell density is 1 * 10
7/ ml; Be sub-packed in frozen pipe, 1ml/ props up, and puts 4 ℃ of refrigerator 1h, changes-20 ℃ of refrigerators again over to and places 1h, changes-40 ℃ of refrigerator 1h again over to, changes liquid nitrogen container again over to and makes a slip of the tongue night, and immerse liquid nitrogen cryopreservation with frozen cell next day.
Induce the ascites legal system in embodiment 3 bodies and be equipped with monoclonal antibody
Get 10 6-8 BALB/C mice in all ages, the only pre-sensitization of abdominal injection whiteruss 0.4ml/ uses hybridoma 2B4 (CGMCC No.5124) with 1 * 10 behind the 7d
6The mouse of the above-mentioned pre-sensitization of amount abdominal injection of individual/(equivalent physiological saline only is diluted to 500 μ l/), observe mouse behind the 10-14d, abdominal distension obviously, be slow in action, put to death when anorexia and hair entanglement and adopt ascites, with syringe ascites is collected in the centrifuge tube, 2000r/min, centrifugal 10min gets the ascites that supernatant is a large amount of monoclonal antibody 2B4, it is standby to put 4 ℃ of refrigerators, and prolonged preservation is then frozen-20 ℃ or-40 ℃.
The evaluation of embodiment 4 monoclonal antibody subclass
Adopt Mouse Monoclonal Antibody Isotyping Kit test kit (Pierce company), concrete steps are as follows with reference to its specification sheets:
(1) take out enzyme plate, each sample needs 6 holes, and cells and supernatant 50 μ l/ holes are added enzyme plate, each sample adds 6 holes, positive control and negative control also are respectively to add the every hole 50 μ l in 6 holes, and every hole adds 50 μ l sample diluents more then, sticks 37 ℃ of incubation 30-40min of shrouding film.
(2) washing plate machine (BIO-TEK company product) washes 5 times, in 6 holes of each sample, add respectively again 6 kinds of ELIAS secondary antibody (IgG1 IgG2a IgG2b IgG3 IgM IgA) each 100 μ l, 6 holes of general positive contrast too, adding on the master drawing or enzyme plate is carried out mark, sticking 37 ℃ of incubation 30-40min of shrouding film.
(3) wash the plate machine washing 5 times, every hole adds developer A and each 50 μ l of developer B respectively, changes 37 ℃ of colour developings of new shrouding film pasting board lucifuge 20-30min, the termination reaction result of determination.
Measurement result, the monoclonal antibody of hybridoma cell strain 2B4 (CGMCC No.5124) secretion is IgG1, κ.
The purifying of embodiment 5 monoclonal antibodies
Carry out the purifying of ascites with HiTrap affinity columns (GE company), step is as follows:
(1) ascites binding buffer (20mM Na
2HPO
4, PH 8-9) and be diluted to 3 times, the filter with 0.22 filters.
The binding buffer of (2) 10 times of column volumes washes pillar, and the bubble on the constant flow pump of draining in the sample pipe, flow velocity are 1mlmin
-1
(3) add the good ascites sample of filtration in above-mentioned (1) with constant flow pump.
(4) the binding buffer with 5-10 times of column volume washs
(5) with the elution buffer of 2-5 column volume (0.1M citric acid, PH4.5-6.0) wash-out monoclonal antibody and be collected in and shift to an earlier date (every pipe adds the 1M Tris-HCl of 60-200 μ l, and pH 9.0) in the ready collection tube
(6) every pipe is got 15 μ l and is carried out SDS-PAGE and analyze its purification effect, and surveys concentration, and concentration is 2.173mg/ml.
The CHARACTERISTICS IDENTIFICATION of embodiment 6 monoclonal antibodies
The titration of step 1 Hybridoma Cell Culture supernatant and monoclonal antibody
Two kinds of method for measuring basically identicals:
Operate according to embodiment 2 steps 2, difference is to add cell conditioned medium to be checked (hybridoma CGMCC No.5124 supernatant) extent of dilution to be respectively: 1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200, extent of dilution was 1: 1000,1: 3000,1: 9000,1: 27000,1: 81000 when survey ascites was tired.With the negative contrast of the serum of non-immune BALB/C mice, rabbit anteserum with anti-JEV resists (with the ordinary method preparation) as positive control (1: 50) more, many anti-corresponding two anti-ly are the staphylococcal protein A,SPA HRP-SPA of horseradish peroxidase-labeled (1: 5000, structure is from Wuhan doctor's moral reagent company) result such as table 1 and table 2, as seen tiring greater than 1: 2500 of hybridoma CGMCC No.5124 supernatant, tiring of monoclonal antibody 2B4 (embodiment 4 purifying) is about 1: 80000.
The titration of the cell conditioned medium of table 1,2B4
The titration of table 2,2B4 monoclonal antibody
To recover behind hybridoma (CGMCC No.5124) cultured continuously two months and the liquid nitrogen cryopreservation, get the cells and supernatant in 5,10,20 generations, survey its antibody titer by case study on implementation 2 step 2 programs with indirect elisa method, with the negative contrast of SP2/0 cell culture fluid.The result is as shown in table 3, and antibody-secreting is stable.
The ELISA antibody titer of table 3, different generation hybridoma supernatants
(1) virus culture
Cultivate the sensitive cells BHK (Shanghai biological company limited of brilliant section) of encephalitis with the DMEM nutritive medium that contains 8% calf serum, outwell nutritive medium after in rolling bottle, growing up to individual layer, after trysinization, blow and beat with suction pipe and to get off to make cell suspension, insert in the 96 porocyte plates, put 37 ℃ 5% CO
2Cell culture incubator is cultivated.Every porocyte length such as second day is to about the 70-80%, outwell nutritive medium, the DMEM of PBS or serum-free or keep liquid (2%DMEM) and wash once pats dry at thieving paper gently, add JEV virus liquid 100 μ l/ holes, establish the hole that do not add poison as the CO of 37 ℃ 5% of negative control
2Cell culture incubator effect 2h.The venom of preventing or cure a disease with the DMEM of PBS or serum-free or keep liquid (2%DMEM) and wash once, blots (preventing crossed contamination) with pipettor, adds and keeps liquid 100 μ l/ holes, puts 37 ℃ 5% CO
2Cell culture incubator is cultivated.
(2) indirect immunofluorescence
Behind the 48h, incline and keep liquid, PBS washing back fixedly 10min of-20 ℃ of cold acetone/4 ℃ straight alcohols discards stationary liquid, and 96 porocyte plates are dried naturally.Monoclonal antibody 2B4 is by 10
-1, 10
-2, 10
-3, 10
-4Four gradient dilutions (original concentration is 2.173mg/ml), the positive (how anti-) contrast and negative (PBS) contrast are established in 50 μ l/ holes, 37 ℃ of incubator effect 45min, PBS washing 3 times is dried.It is anti-to add 50 μ l/ hole sheep anti mouse fluorescence two, 37 ℃ of incubator effect 45min, and observations under the inverted fluorescence microscope is put in PBS washing 3 times.The result judges: the endochylema that can be observed positive cell in the positive cell hole has typical specificity bright green fluorescence, and no specificity green fluorescence in the cell hole of feminine gender and blank.As Fig. 3, with reference to yin and yang attribute contrast, 10
-1With 10
-2The time 2B4 fluorescence more intense.
According to embodiment 1 step 1, with the JEV cell toxicant collected through 3 multigelations, ultrasonic disruption, preliminary centrifugal purification, carry out the 15%SDS-PAGE electrophoresis, do not infect the negative contrast of bhk cell lysate of JEV, in Bio-Rad electrotransfer system, the gel protein band is transferred on the nitrocellulose filter according to a conventional method, spend the night in 4 ℃ of shaking tables (TY-80S type decolorization swinging table reaches biotechnology development company product for Nanjing University south) sealing with confining liquid (TBS that contains 5% skim-milk), adding is by the monoclonal antibody 2B4 of embodiment 3 preparations and purifying, room temperature is put shaking table reaction 2 hours, TBST (PH7.5,0.05mol/L Tris-HCl, 0.15mol/L NaCl, 0.05%Tween-20) washing is 3 times, each 10min.The sheep anti-mouse igg that adds the HRP mark then, mother liquor are confining liquid, and room temperature shaking table reaction 2h washs 3 times (method as above).At last, in a cleaning plate, prepare colour developing liquid 5mL (now with the current) according to DAB colouring reagents box, to wash good nitrocellulose filter then puts into wherein, shake gently, namely use aquae destillata rinsing nitrocellulose filter with termination reaction in case the band colour developing is clear, taking pictures after the taking-up, it is standby to preserve.As shown in Figure 4,2B4 and JEV have stronger and very single reaction, and do not react with the bhk cell lysate.
Step 5 flow cytometry monoclonal antibody 2B4 under field conditions (factors) with bhk cell and infect JEV bhk cell in conjunction with situation
With reference to the cultural method in the present embodiment step 3 (1), place six orifice plates to grow up to individual layer bhk cell, approach when covering with, DMEM with serum-free washes one time, with the amount inoculation JEV in the every hole of 500 μ l/, 37 ℃ of incubator effect 1.5-2h are contrast (2 groups) with the cell that does not process.Add 1ml Digestive system (PBS+0.02%EDTA) then and place 10min, cell is blown out suspension, centrifugal (1500r/8min), remove supernatant, it is resuspended to add 200 μ lPBS, centrifugal again, abandon and dilute good primary antibodie with 200 μ l behind the supernatant (2B4 is 21.73 μ g/ml, mother liquor is PBS) carry out resuspended (blank does not add), incubated at room 1h washes twice with PBS according to above-mentioned washing methods, (it is anti-that negative control adds fluorescence two to add sheep anti mouse fluorescence two anti-incubated at room 40min then, and blank does not add), after the PBS washed twice, (BD Biosciences) observes with flow cytometer.
As Fig. 5, according to blank and negative control, after bhk cell infected JEV, the combination rate of monoclonal antibody 2B4 significantly rose, and illustrated that monoclonal antibody 2B4 can be combined with JEV under state of nature.
The neutralization activity of embodiment 7 monoclonal antibody 2B4
The neutralization activity of step 1 in cell experiment
(1) neutralization experiment
The monoclonal antibody 2B4 of embodiment 5 purifying is done 3 gradient dilutions (10
-1, 10
-2, 10
-3) be diluted to 50 μ g, and mix 4 ℃ of overnight incubation with the JEV of isopyknic 100TCID50.Simultaneously, with reference to the cultural method in embodiment 5 steps 4 (1), place 96 orifice plates to grow up to individual layer bhk cell, the nutritive medium that inclines is washed one time with the DMEM of serum-free, adds above-mentioned reaction solution (100 μ l/ hole), each gradient repeats 10 times, places 37 ℃ of effect 1.5-2h.Then, outwell reaction solution, wash one time with the DMEM of serum-free, add and keep liquid (DMEM that contains 2% calf serum, 100 μ l/ holes).Observation of cell upgrowth situation after 6-8 days, occur cell rounding or irregular, come off, situation such as cytoadherence, monolayer cell cavity then judges cell generation pathology.Calculate the pathology per-cent of each gradient, plot Fig. 6.
As Fig. 6, as seen dilute 10
-3Beginning among the monoclonal antibody 2B4 and reduced activity, therefore proves that it has certain neutralization activity.
(2) neutralization experiment-indirect immunofluorescence
Monoclonal antibody 2B4 is done 4 gradient dilutions (10
-1, 10
-2, 10
-3, 10
-4Original concentration is 2.173mg/ml), each gradient repeats 4 times, blank is set, according to the step operation of neutralization experiment in the step 1, according to the operation of embodiment 4 steps 4, negative control (cell that virus infection is crossed and only add fluorescence two anti-) is set behind the 72h, with how anti-(PAB) positive contrast of neutrality, difference is that the primary antibodie extent of dilution is 10
-2Result such as Fig. 7, having the hole of 1/2 quantity that the judgement fluorescence intensity of fluorescence is arranged is 1, it is 2 that there is the judgement fluorescence intensity of fluorescence in hole more than 3/4, the judgement fluorescence intensity that fluorescence is arranged below 1/4 is 0, therefore, be 0 for the how anti-PAB fluorescence intensity of neutrality, and the negative control fluorescence intensity is 2 always always, comparatively speaking, be 10 at extent of dilution
-1, 10
-2The time 2B4 have neutralization active, this with in consistent with result of experiment.
The neutralization activity of step 2 in experimentation on animals
The 2B4 that will contain 50mg monoclonal antibody albumen mixes the back and spends the night 4 ℃ of reactions with the JEV cell toxicant (according to the amount of LD50 preliminary experiment gained) of 500 μ l, injection BALB/c mouse (9-12 age in days), and 10 one group, every of 500 μ l/, and control group is set, only inject JEV.Result such as table 4, the survival number of mouse obviously improves, and the death time also postpones, and illustrates that 2B4 has certain neutralization activity under state of nature, gets a good chance of carrying out developing vaccines research.
In table 4, the animal and the experiment
Annotate: the death time refers to from injecting the back to the pitch time that begins to take place death
The application of embodiment 8 monoclonal antibody 2B4 detection of cancerous encephalitis B suckling mouse brain spinal fluids
(1) gets suckling mouse (3 days) cerebral tissue of attacking the strong malicious intercurrent disease of encephalitis and dying, get same batch simultaneously not attack the good suckling mouse brain tissue of poison and growth conditions.After clean with normal saline flushing, adding 1ml fully grinds, the centrifugal 10min of 3000r/min, get supernatant, filter with 0.22 filters (aseptic technique), standby as extracting solution, the final sample of attacking after the dead mouse brain organized processing of the strong malicious intercurrent disease of encephalitis is labeled as BS-V, and the final sample of not attacking after the good mouse brain organized processing of poison and growth conditions is labeled as BS.
(2) according to the step of embodiment 6 step 3 indirect immunofluorescences, BS-V made 10 times gradient dilution (10
-1, 10
-2, 10
-3, 10
-4), BS does not dilute, and makes 1.5-2h with the sense of BHK-21 cell, and monoclonal antibody is by 10
-2Add 50 μ l/ holes, all the other steps are identical.
Result such as Fig. 8, BS-V is in gradient 10
-1, 10
-2, 10
-3Stronger fluorescence (the microscope multiple turns down to observe more cell) is arranged, and BS is then consistent with negative control.This experimental results show that monoclonal antibody 2B4 can detect the existence of JEV in the cerebrospinal fluid under clinical condition, thereby for further detection method, detection kit and the vaccine etc. of research and development encephalitis b virus are laid a good foundation, for clinical diagnosis and the treatment of encephalitis important application prospects is arranged.
Claims (3)
1. secrete the hybridoma cell strain 2B4 of the monoclonal antibody of anti-encephalitis b virus, on August 3rd, 2011 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.5124.
2. the monoclonal antibody of an anti-encephalitis b virus is produced by the described hybridoma cell strain of claim 1.
3. the application of the described monoclonal antibody of claim 2 in the detection reagent of preparation detection encephalitis b virus.
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